It is well known that the properties of proteins may be modified by covalently conjugating groups to said proteins. Such conjugation generally requires some functional group in the protein to react with another functional group in the compound to be conjugated to the protein. Typically, amino groups, such as the N-terminal amino group, the ∈-amino group in lysines or the cysteine thiol group have been used in combination with a suitable acylating or alkylating reagent.
It is often desired to conjugate at specific sites, because the biological activity of a chemically modified protein will depend on the site of modification, and this is referred to as regioselective conjugation. Regioselective acylation of growth hormones, such as for instance human growth hormone (hGH) is, however, difficult because this protein contains nine lysine residues of similar reactivity, and mixtures of products usually result. The single components of these mixtures are difficult to isolate, and will therefore usually be obtained in low yield and purity only.
One strategy to attain regioselective conjugation of a property-modifying group to a protein consists in generating a functional group in the protein, which cannot be found in the natural proteinogenic amino acids. Such a unique functional group may, for instance, be an aldehyde or ketone, an alkyne, or an azide.
In particular protein-derived aldehydes have often been used for the conjugation of property-modifying groups to proteins. This conjugation can be achieved by treating the protein-derived aldehyde with an alkoxylamine (R—O—NH2) to form an oxime, with hydrazine-derivatives to form hydrazones, or with 2-aminoethanethiols to form thiazolidines (see for instance Zhang and Tam, Analytical Biochemistry 233, 87-93 (1996); Zatsepin et al., Bioconjugate Chemistry 13, 822-830 (2002)). All these conjugating functional groups are hydrolytically unstable, and may be hydrolyzed by the treatment with acids or bases (Shao and Tam, J. Am. Chem. Soc. 117, 3893-3899 (1995)) at much higher rates than normal peptide bonds. For this reason, the above mentioned conjugates will slowly decompose in aqueous solution, what limits their utility.
Gaertner et al (Bioconjugate Chem. 7, 38-44 (1996)) disclose a method for conjugating Polyethylene glycol (PEG) to IL-8, G-CFS and IKL-1ra by generating an aldehyde at the N-terminus of these proteins, followed by reaction with an alkoxyamine-functionalized PEG. The aldehyde is generated at the N-terminus either by oxidation with periodate if the N-terminal amino acid residue is serine or threonine, or by metal catalyzed oxidative deamination if the N-terminal amino acid residue is different from serine.
N-terminal serine-extended human growth hormone, Ser-hGH, is disclosed as SEQ ID No. 66 in WO 04/007687. This application relates to multimeric forms of for instance hGH with improved properties.
US 20040127417 describes the reductive alkylation of native growth hormone with a PEG-derived aldehyde. In said procedure the slight difference in basicity between the lysine side-chain amino groups and the N-terminal amino group is exploited: under acidic conditions imine formation with the less extensively protonated N-terminal amino group is faster than imine formation with the lysine side-chains, so that a regioselective PEGylation of the N-terminal amino acid can be achieved.